Neuroactive substances play important roles as neuromodulators as well as neurotransmitters. One of them, acetylcholine (ACh), is known to be present in taste receptor cells and in nerve fibers innervating taste buds, and ACh agonists improve taste sensation in patients with taste disorders. However, mechanisms underlying physiological functions of ACh in taste receptor cells are not understood. In non-gustatory systems, five subtypes of muscarinic ACh receptors have been identified. I have shown that ACh induces increases in intracellular Ca2+ in taste receptor cells via activation of muscarinic receptors, and that positive immunoreactivity for the M1 subtype of muscarinic receptor is present in both apical and basolateral regions of taste receptor cells. Therefore, I hypothesize that muscarinic ACh receptors may play a role in both taste modulation and taste transduction. Similarly, glutamate receptors function as taste receptors for "umami" taste, in addition to their well known role in synaptic transmission and neuromodulation. In this proposal, I intend to further study the physiological function of ACh and its modulatory effect on taste responses, specifically bitter taste transduction. In addition, ACh receptors at apical membrane of taste receptor cells may function as taste receptors. Three specific aims are: (1) characterize muscarinic ACh receptors in taste cells with pharmacological agents and mice deficient for a specific receptor subtype; (2) determine the second messengers that mediate ACh-induced modulation of bitter responses; and (3) determine whether muscarinic receptors function as taste receptors. For these aims, experiments will be performed using Ca2+ imaging and in situ nerve recording techniques with three animal models: mouse, rat, and mudpuppy. Each species has unique advantages for particular experimental approaches, and all species appear to utilize the same signal transduction mechanisms, in response to ACh. Results from the proposed experiments will provide significant new information about muscarinic ACh receptors in taste buds and their role in taste modulation and perception. In addition, the proposed studies will broaden our understanding about bitter detection mechanisms. This project is expandable to investigate the physiological role of other neuroactive substances in taste cells in future experiments.